Chakraborty, S.

ChemSusChem 2021, 14, 2237-2246, 10.1002/cssc.202100122

Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI-peptide and protonation of the NiII−H− intermediate (Ni−R) by a Cys (pKa≈6.4) to produce H2.

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.

Lu, Y.

Angew. Chem. Int. Ed. 2012, 51, 5589-5592, 10.1002/anie.201201981

Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.

Lu, Y.

Science 2018, 361, 1098-1101, 10.1126/science.aat8474

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 11570-11573, 10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Tezcan, F.A.

Science 2014, 346, 1525-1528, 10.1126/science.1259680

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

Hayashi, T

Chem. Commun. 2011, 47, 8229, 10.1039/c1cc11157d

The diiron carbonyl cluster is held by a native CXXC motif, which includes Cys14 and Cys17, in the cytochrome c sequence. It is found that the diiron carbonyl complex works well as a catalyst for H2 evolution. It has a TON of ∼80 over 2 h at pH 4.7 in the presence of a Ru-photosensitizer and ascorbate as a sacrificial reagent in aqueous media.

DeGrado, W.F.

Nat. Chem. 2012, 4, 900-906, 10.1038/NCHEM.1454

De novo proteins provide a unique opportunity to investigate the structure–function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

DeGrado, W.F.; Lombardi, A.

Nat. Chem. Biol. 2009, 5, 882-884, 10.1038/nchembio.257

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

Reetz, M.T.

Angew. Chem. Int. Ed. 2010, 49, 5151-5155, 10.1002/anie.201002106

Guided by nature: A designed binding site comprising the His/His/Asp motif for CuII complexation has been constructed in a robust protein by site‐specific mutagenesis (see picture). The artificial metalloenzyme catalyzes an enantioselective Diels–Alder reaction.

Gao, X.; Zhao, L.

Sci. Adv. 2020, 6, 10.1126/sciadv.abb1421

Metalloenzymes are promising anticancer candidates to overcome chemoresistance by involving unique mechanisms. To date, it is still a great challenge to obtain synthetic metalloenzymes with persistent catalytic performance for cancer-specific DNA cleavage and operando imaging. Here, an artificial metalloenzyme, copper cluster firmly anchored in bovine serum albumin conjugated with tumor-targeting peptide, is exquisitely constructed. It is capable of persistently transforming hydrogen peroxide in tumor microenvironment to hydroxyl radical and oxygen in a catalytic manner. The stable catalysis recycling stems from the electron transfer between copper cluster and substrate with well-matched energy levels. Notably, their high biocompatibility, tumor-specific recognition, and persistent catalytic performance ensure the substantial anticancer efficacy by triggering DNA damage. Meanwhile, by coupling with enzyme-like reactions, the operando therapy effect is expediently traced by chemiluminescence signal with high sensitivity and sustainability. It provides new insights into synthesizing biocompatible metalloenzymes on demand to visually monitor and efficiently combat specific cancers.

Kehl-Fie, T.E.; Waldron, K.J.

Nat. Commun. 2020, 11, 10.1038/s41467-020-16478-0

AbstractAlmost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.

Utschig, L.M.

Chem. Commun. 2015, 51, 10628-10631, 10.1039/c5cc03006d

Long-lived charge separation facilitates photocatalytic H2 production in a mini reaction center/catalyst complex.

Fujieda, N.; Itoh, S.

Chem. - Asian J. 2012, 7, 1203-1207, 10.1002/asia.201101014

Teaching metalloenzymes new tricks: An artificial type III dicopper oxidase has been developed using a hydrolytic enzyme, metallo‐β‐lactamase, as a metal‐binding platform. The triple mutant D88G/S185H/P224G redesigned by computer‐assisted structural analysis showed spectroscopic features similar to those of type III copper proteins and exhibited a high catalytic activity in the oxidation of catechols under aerobic conditions.

Borovik, A.S.; Hendrich, M.P.; Moënne-Loccoz, P.

J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Hartwig, J.F.

J. Am. Chem. Soc. 2022, 144, 883-890, 10.1021/jacs.1c10975

The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C–H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.

Apfel, U.-P.; Happe, T.; Kurisu, G.

Chem. Sci. 2016, 7, 959-968, 10.1039/C5SC03397G

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.

Dömötör, O.; Enyedy, É.A.

J. Biol. Inorg. Chem. 2019, 24, 703-719, 10.1007/s00775-019-01683-0

Various half-sandwich ruthenium(II) arene complexes and rhodium(III) arene complexes have been intensively investigated due to their prominent anticancer activity. The interaction of the organometallic complexes of Ru(η6-p-cymene) and Rh(η5-C5Me5) with human serum albumin (HSA) was studied in detail by a combination of various methods such as ultrafiltration, capillary electrophoresis, 1H NMR spectroscopy, fluorometry and UV–visible spectrophotometry in the presence of 100 mM chloride ions. Binding characteristics of the organometallic ions and their complexes with deferiprone, 2-picolinic acid, maltol, 6-methyl-2-picolinic acid and 2-quinaldic acid were evaluated. Kinetic aspects and reversibility of the albumin binding are also discussed. The effect of low-molecular-mass blood components on the protein binding was studied in addition to the interaction of organorhodium complexes with cell culture medium components. The organometallic ions were found to bind to HSA to a high extent via a coordination bond. Release of the bound metal ions was kinetically hindered and could not be induced by the denaturation of the protein. Binding of the Ru(η6-p-cymene) triaqua cation was much slower (ca. 24 h) compared to the rhodium congener (few min), while their complexes interacted with the protein relatively fast (1–2 h). The studied complexes were bound to HSA coordinatively. The highly stable and kinetically inert 2-picolinate Ru(η6-p-cymene) complex bound in an associative manner preserving its original entity, while lower stability complexes decomposed partly or completely upon binding to HSA. Fast, non-specific and high-affinity binding of the complexes on HSA highlights their coordinative interaction with various types of proteins possibly decreasing effective drug concentration.

Emerson, J.P.

J. Biol. Inorg. Chem. 2013, 18, 595-598, 10.1007/s00775-013-1009-1

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu2+ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (K a approximately 5 × 1012 and 1 × 1010), and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

Albrecht, M.; Paradisi, F.

Angew. Chem. Int. Ed. 2018, 130, 10837-10842, 10.1002/ange.201807168

Im Tausch gegen NHC: Die Einfügung eines N‐heterocyclischen Carbenliganden (grün/blau) als Ersatz für His in das aktive Zentrum des Redoxenzyms Azurin rekonstituiert das T1‐Kupferzentrum. Der resultierende Komplex ist spektroskopisch kaum unterscheidbar von der N‐Bindung von His oder N‐Methylimidazol, senkt aber signifikant das Reduktionspotential des Kupferzentrums und erleichtert dadurch Elektronentransferprozesse.

Ward, T.R.

Org. Biomol. Chem. 2015, 13, 5652-5655, 10.1039/c5ob00428d

We report an efficient artificial metathesase which combines an arylsulfonamide anchor within the protein scaffold human carbonic anhydrase II.

Kuhlman, B.

Biochemistry 2012, 51, 3933-3940, 10.1021/bi201881p

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein–protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 105 and a kcat/KM of 630 M–1 s–1 and 4-nitrophenyl phosphate with a rate acceleration of 104 and a kcat/KM of 14 M–1 s–1. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein–protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Lu, Y.

J. Am. Chem. Soc. 2006, 128, 6766-6767, 10.1021/ja058822p

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV−vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO·mol CuBMb-1·min-1, close to 3 mol NO·mol enzyme-1·min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV−vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV−vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron−proximal histidine toward a five-coordinate key intermediate in NO reduction.

Apfel, U.-P.; Happe, T.

Dalton Trans. 2017, 46, 16947-16958, 10.1039/C7DT03785F

Combination of biological and chemical methods allow for creation of [FeFe]-hydrogenases with an artificial synthetic cofactor.

Baker, D.

Nat. Chem. Biol. 2012, 8, 294-300, 10.1038/NChemBio.777

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (kcat/Km) of ∼104 M−1 s−1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the RP isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

Makhlynets, O.V.

J. Inorg. Biochem. 2020, 212, 111224, 10.1016/j.jinorgbio.2020.111224

Metalloproteins constitute nearly half of all proteins and catalyze some of the most complex chemical reactions. Recently, we reported a design of 4G-UFsc (Uno Ferro single chain), a single chain four-helical bundle with extraordinarily high (30 pM) affinity for zinc. We evaluated the contribution of different side chains to binding of Co(II), Ni(II), Zn(II) and Mn(II) using systematic mutagenesis of the amino acids that constitute the primary metal coordination and outer spheres. The binding affinity of proteins for metals was then measured using isothermal titration calorimetry. Our results show that both primary metal coordination environment and side chains in the outer sphere of UFsc are highly sensitive to even slight changes and can be adapted to binding different 3d metals, including hard-to-tightly bind metal ions such as Mn(II). The studies on the origins of tight metal binding will guide future metalloprotein design efforts.

Barker, P.D.; Boss, S.R.

Angew. Chem. Int. Ed. 2021, 60, 10919-10927, 10.1002/anie.202015834

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6-arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

Ueno, T.; Watanabe, Y.

J. Am. Chem. Soc. 2008, 130, 10512-10514, 10.1021/ja802463a

We report the preparation of organometallic Pd(allyl) dinuclear complexes in protein cages of apo-Fr by reactions with [Pd(allyl)Cl]2 (allyl = η3-C3H5). One of the dinuclear complexes is converted to a trinuclear complex by replacing a Pd-coordinated His residue to an Ala residue. These results suggest that multinuclear metal complexes with various coordination structures could be prepared by the deletion or introduction of His, Cys, and Glu at appropriate positions on protein surface.

Fujieda, N.; Itoh, S.

Angew. Chem. 2020, 132, 7791-7794, 10.1002/ange.202000129

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded β-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 4594-4597, 10.1021/ja5109936

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.